Non-protein-coding (nc)RNAs are undergoing a renaissance in biology due to the identification of myriad new sequences and functions. To keep pace with findings, new progress is needed to deduce the mechanisms of action of representative ncRNAs in order to elucidate the broad principles that impart function to molecules of emergent importance in human health. Previously, we discerned key structure- function relationships for two ncRNAs: (i) the hairpin ribozyme (HPRZ), a representative of the small RNA enzyme family, and (ii) a metabolite-sensing riboswitch that responds to preQ1, a precursor to anticodon modifications. For the HPRZ, we synthesized new conformational mimics of the reaction coordinate, and used them to pinpoint a key imino group at Ade38 indispensible for catalysis. We further hypothesized that specific conformational changes along the reaction coordinate perturb the microscopic pKa of Ade38 to impart vital properties for acid/base catalysis or electrostatic stabilization of the transition state. These possibilities represent key mechanistic distinctions in the field with the potential to inform catalytic precedents adopted by universally conserved RNA enzymes. In another milestone, we determined the structure of a novel (class I) preQ1 riboswitch to reveal how a bacterial-specific metabolite is recognized. This work also described the unique mode by which the riboswitch sequesters a nearby ribosome binding site (RBS) leading to mRNA translational arrest. Although the manner of preQ1 recognition by this class of riboswitches appears conserved, RBS sequestration appears to occur by divergent means. This raises questions of how other preQ1 riboswitches bury gene expression signals, which has implications for antibiotic discovery and targeting. The central hypothesis of this proposal is that ncRNAs of specialized function can extend the chemical repertoire of bases by adopting novel 3-D folds that form unique microenvironments. Our two-year specific aims are: (1) to evaluate the spatial relationships between our known structures of the HPRZ reaction coordinate and the ability of these defined states to perturb the pKa of Ade38 toward neutrality. High-resolution structures are already in hand that will be related directly to pKa measurements on single crystals made by Raman spectroscopy. New preliminary data indicate a single Ade of HPRZ crystals has its pKa shifted from 4.35 to 5.8, which is proof of principle for our approach;and (2) to determine the molecular principles that govern how phylogenetically disparate riboswitches bind the preQ1 metabolite leading to sequestration of essential gene expression signals, such as the RBS. Preliminary data are available from crystallographic, small angle scattering and biochemical approaches. A long-term benefit is that the results will provide new insight into the complex functions of emergent catalytic and gene-regulatory ncRNAs pertinent to public health.